r goal is to identify cellular and molecular mechanisms that determine the pattern in which neurons are generated in the zebrafish neural plate. We are using a combination of cellular, molecular and genetic approaches to identify mechanisms involved in this process. We are investigating a neurogenic mutant, mindbomb (mib), which is characterized by an over-production of early neurons. Previous observations suggest a defect in the neurogenic gene pathway, which mediates lateral inhibition and limits the number of neurons produced, in these mutants. Our linkage analysis, however, shows that mib does not appear to be linked to many of the known zebrafish neurogenic gene homologues including Delta A, B and D and Notch 1b and Notch3. We are in the process of identifying closely linked CA repeat length polymorphisms in order to put mib on a reference linkage map for zebrafish and to eventually clone the gene. Functionally, we have shown that mib mutants are responsive to the effects of activated Notch, which reduces neurons in both wild type and mib mutant embryos. We have also shown that ectopic expression of a proneural gene, neurogenin, leads to an unusually high density of ectopic neurons in mutant embryos, consistent with a failure of lateral inhibition in the mutants. Our functional analysis of mib mutants is continuing with a) analysis of the effects of ectopic expression of other genes in the neurogenic gene pathway and b) transplantation experiments to determine if mib mutants are defective in sending or receiving signals that limit the number of neurons. Mib mutant embryos are also characterized by aberrant tissue boundaries in the rhombomeres and somites. To investigate the cellular basis of these defects we are generating time-lapse movies to compare cellular rearrangements in wild type and mutant mib embryos during somitogenesis with scanning confocal microscopy. At this stage we have developed movies showing the formation of well-defined boundaries in wild type embryos using this technique. We also have seen poorly defined boundaries in the mutants." " We are now in the process of analyzing the rearrangements of cells in the mutants to identify differences in tissue reorganization in domains preceding the formation of the boundaries. We are also examining the function of zebrafish homologues of the neurogenic gene Notch and some of its ligands in early neurogenesis. Specifically, we are investigating the role of Notch3 I in early neurogenesis. Notch3 is expressed during gastrulation in a dynamic pattern in tissue condensations that presage formation of the neural plate. Consistent with an early role in influencing the formation of the neural plate, we have shown that ectopic expression of an activated form of Notch3 alters the shape of the neural plate. We are investigating how activity of Notch3 influences this early step in neurogenesis by affecting cell fate and/or by affecting morphogenetic movements during the formation of the neural plate. Previously we used an early marker for neurons; the elavC related gene or HuC to identify mutants with aberrant patterns of neurogenesis. In this "insitu" based screen we identified mutants in which ectopic neurons skirt the caudal neural plate. In the past year we have recovered four of these lines and have begun functional analysis of the "skirt" mutants. Our complementation analysis suggests that the "skirt" mutants belong to at least two complementation groups. Skirt mutants suggest that the caudal neural plate is a domain where neurons have the potential to form but inhibitory interactions normally prevent neurons from forming. In the mutants, neurons may appear at the caudal boundary because the inhibitory activity is lost. The potential for making neurons in the caudal neural plate of embryos is also revealed when the proneural gene, neurogenin, is ectopically expressed. Neurogenin RNA injections lead to ectopic neurons in specific domains in the ectoderm where it is easier to make neurons; one of these domains is the caudal neural plate. Our studies are now aimed at revealing why the potential for making neurons is revealed in the skirt mutants. Our analysis of early neurogenesis suggests that cells are selected to become neurons when dynamic cell rearrangements are taking place. To understand how cells selected to become neurons come to occupy particular positions it is necessary to track the movement neurons in the developing embryo. In collaboration with Dr. Huh from Kyungpook National University in Korea we are generating a transgenic zebrafish line in which Green Fluorescent Protein is expressed in embryos under the control of the HuC promoter. These fish will have fluorescent neurons that will be visible in live embryos from the neural plate stage onward. This transgenic line will provide a powerful tool for analysis of the movement of early neurons and will facilitate analysis and identification of neurogenesis mutants in the future. Our analysis of the cellular, molecular and genetic mechanisms that determine the pattern of early neurons will allow us to understand how neurons are made in the right number and location in the nervous system of vertebrates. These studies will lead to insights about human diseases and birth disorders characterized by an aberrant distribution of neurons. Molecular mechanisms that determine the spatial distribution of cells in the nervous system are also used in other organ systems." " Understanding how a simple pattern of neurons is generated in zebrafish will help us learn how cell-cell interactions lead to the self-organization of tissue heterogeneity in the developing embryo."